avidin biotin complex amplification  (Vector Laboratories)

Journal: PLOS ONE

Article Title: Whole-body analysis of TRPML3 (MCOLN3) expression using a GFP-reporter mouse model reveals widespread expression in secretory cells and endocrine glands

doi: 10.1371/journal.pone.0278848

Figure Lengend Snippet: Immunofluorescence images using F4/80 antibody as macrophage marker (A) in 10 μM lung cryosections or (B) on cells isolated by bronchoalveolar lavage. Images clearly show co-localization of τGFP and F4/80 confirming TRPML3 expression in lung macrophages. (C) Trpml3 levels in lung increase from birth to adult. Trpml3 detected by RT-qPCR and normalized as in . Levels are displayed relative to normalized Trpml3 in E18 lung. (D, E) In situ hybridization on sections of neonatal (P2) lungs from Trpml3 +/+ mice shows a pattern of scattered positive cells (arrowheads) detected by both 5’ Trpml3 (B) and 3’ Trpml3 (C) probes. (F-K): Immunohistochemistry using nonfluorescent detection (ABC+DAB) shows NT and CT1 antibodies specifically label scattered cells (arrowheads) in sections of adult (P48) Trpml3 +/+ (F-I) but not Trpml3 -/- lungs (J, K). (L, M): Co-immunohistochemistry on sections of adult (P48) Trpml3 +/+ lungs indicates that NT colabels F4/80 positive macrophages. Scale bars indicate 50 μm in D, E, F, G, J, K and 10 μm in H, I, L, M.

Article Snippet: Where noted, we used the ABC/DAB (avidin-biotin complex with diaminobenzidine reaction) signal amplification system (Vector).

Techniques: Immunofluorescence, Marker, Isolation, Expressing, Quantitative RT-PCR, In Situ Hybridization, Immunohistochemistry

Journal: PLOS ONE

Article Title: Whole-body analysis of TRPML3 (MCOLN3) expression using a GFP-reporter mouse model reveals widespread expression in secretory cells and endocrine glands

doi: 10.1371/journal.pone.0278848

Figure Lengend Snippet: (A) Overview image of a longitudinal section of an adult kidney. RP, renal pelvis. RC, renal cortex. CD, collecting ducts. (B) Magnified image of the renal medulla with collecting ducts, which open into the renal pelvis. Cells of the collecting ducts seem to express τGFP. (C) Zoomed image of the renal cortex revealing renal tubules, of which some contain τGFP+ cells. (D) Assembled montage showing in situ hybridization on section of neonatal Trpml3 +/+ kidney using 5’ Trpml3 probe showing Trpml3 expression in select tubes. ( E, F) Immunohistochemistry on adjacent Trpml3 +/+ neonatal kidney sections using NT (E) and aquaporin 1 (F) antibodies shows that NT does not label the same tubes as aquaporin 1. (G-J) Co-immunohistochemistry shows that TRPML3 amino-terminal antibody NT colabels aquaporin 2 positive tubes in Trpml3 +/+ (G, H) but not Trpml3 -/- (I, J) neonatal kidney. (K-P) Magnified, high resolution optical sections taken perpendicular to a single tube within the Trpml3 +/+ section photographed for G and H do not show a high degree of colocalization between TRPML3-positive and aquaporin 2-positive vesicles. (Q, R) Immunohistochemistry using nonfluorescent detection (ABC+DAB) on adult kidney sections reveal that NT also labels tubes in Trpml3 +/+ (N, insets) but not Trpml3 -/- (O) adult kidney. Insets in N show high resolution of select tube cross sections (boxes) showing large vesicles (arrowheads). Ages of tissue used are: P2 (D), P13 (G-P), P7 (E, F), and P48 (Q, R). Scale bars indicate 500 μm (D), 50 μm (E-J, Q, R), 10 μm (insets on Q) and 2 μm (K-P).

Article Snippet: Where noted, we used the ABC/DAB (avidin-biotin complex with diaminobenzidine reaction) signal amplification system (Vector).

Techniques: In Situ Hybridization, Expressing, Immunohistochemistry